1. Field of the Invention
The present invention relates to a nanochannel system including a nanofluidic device for rapid DNA sequencing with single-base resolution and single nanoparticle characterization based on electron tunneling, and in particular, to a method of fabrication of such a nanochannel by means of the combination of microelectromechanical system (MEMS) microfabrication techniques, atomic force microscopy (AFM) nanolithography, and focused ion beam (FIB).
2. Description of Related Art
Microfluidic devices have become more accepted as a method for rapid biomolecule detection, analysis, and characterization. With an increasing interest in nanotechnology and its many applications, nanofluidic devices are a new area of focus for both academic research and industry. Nanofluidics is often defined as the study and application of fluid flow in and around nanoscale objects [1]. Such devices are currently being investigated in hopes of revolutionizing the conventional method to sequence the entire human genome. Deoxyribonucleic acid (DNA) contains the genetic code of all living organisms, and it is apparent that obtaining its code rapidly and inexpensively would generate a plethora of benefits to our society. Advancements in DNA sequencing methodologies could potentially revolutionize medical research and provide new avenues of exploration for genetics, bioinformatics, molecular biology, biotechnology, and other relative fields.
Ever since the Human Genome Project was launched in October of 1990, there have been drastic improvements in human genome sequencing research and development [2]. In January of 2008, the 1000 Genomes Project was launched as an international research effort to learn more about DNA sequencing and disease detection and to successfully sequence over 1000 human genomes. By October of 2012, 1,092 human genomes were sequenced around the world, including the US, China, Japan, Kenya, Finland, and Peru [3]. Although this project successfully demonstrated the capability of sequencing over 1,000 human genomes and provided researchers with disease detection information, it did not advance the DNA sequencing process any further. This project cost about $120M, meaning that each genome cost approximately $109K and took about 42 hours [4]. Meanwhile, extensive research has been conducted in order to make human genome sequencing more affordable and faster. The current push is for a rapid, label free method that can sequence the entire genome within a few hours at a cost less than $1,000 [5]. According to the National Human Genome Research Institute, the current state of the art allows the entire human genome to be sequenced for approximately $8K and takes anywhere from 10 hours to 2 days. These costs can be misleading, however. For instance, they do not take into account the equipment costs that fall between $400-500K, the facility costs, the interpretation program cost, and other additional sequencing costs [6, 7]. Tremendous progress (≈$10M to $10K per human genome) has been made in the last decade, but in order to make human genome sequencing a routine medical procedure, prices and sampling times must continue to decline to around $1,000 and less than 2 hours.
If the cost-to-sequencing continues to reduce in this manner, then human genome sequencing may become the new standard in healthcare. For instance, healthcare professionals would have access to the entire genome sequence of their patients and, for the first time, would have the possibility to provide medications based on their patients' individual genetic makeup. Individual analyses of the human genome can be used to predict future diseases and help minimize the consequences associated with them. In order for this type of industry to exist commercially, there are still some improvements that need to be made.
Nanopore Sequencing: One of the groundbreaking approaches for solid-state based rapid genome sequencing is the nanopore method. In this label-free approach, single stranded DNAs are translocated through a nanoscale opening as a result of an external electric field [8]. Individual nucleotides are sensed due to their ability to block the monitored current through the nanopore [9]. In nature, DNA is composed of four different bases: adenine (A), cytosine (C), guanine (G), and thymine (T). Theoretically, the four different bases will block a different amount of current and, therefore sequencing is possible.
The central problem with this approach is the high translocation speed of the DNAs, resulting in a difficulty to achieve single-base resolution. Typical translocation speeds have been recorded between 0.5-30 mm/s and as high as 5 cm/s, which is too quick for high-resolution signal sampling [10-13]. One possible solution to the high translocation speeds is to induce a magnetic field that opposes the electric field, resulting in a more accurate readout [14]. Other approaches have included increasing the fluid viscosity, DNA trapping, and voltage regulation [12, 15-17]. Another possible solution is to pull the DNAs through a nanochannel that is at least 3 orders of magnitude longer than a nanopore. A nanochannel is essentially an elongated nanopore, which fundamentally embodies a larger drag force that can ultimately slow down the DNA translocation. In addition, recent publications suggest that a nanochannel with embedded electrical sensors can detect single DNA bases and eventually sequence the human genome [18]. Such devices will use tunneling current as opposed to blockage current as the sensing mechanism. Nanochannels provide several benefits for biomolecule characterization, but they can be challenging to fabricate.
Nanochannel Techniques: Nanochannels are defined as fluid conduits with at least one minimum dimension from <1 nm to 1000 nm [19]. Typical nanochannels are classified as either 1D or 2D, depending on how many dimensions of the channel fall within nanoscale range. Previously, nanochannels have been fabricated through several different methods, such as bulk nanomachining, surface nanomachining, nanoimprint lithography, and direct nanolithography [9, 20, 21]. The bulk nanomachining process creates features out of the body or bulk of a wafer. Trenches are often created by selective patterning and vertical ion plasma etching. These trenches are sealed by a conformal deposited film to create subsurface or buried channels [22]. Scanning electron microscope (SEM) images of bulk machining and nanoimprinting nanochannels are shown in references 23 and 24 cited herein. [23, 24]. Surface nanomachining differs from bulk mainly due to the fact that surface machined nanochannels are created from the removal of a sacrificial layer. This method does not require the bulk wafer to be etched away. Instead, the nanochannel is located on the surface of the wafer. Nanoimprinted nanochannels are formed by a stamping procedure where a mold with nanoscale features is pressed against a wafer covered with photoresist (PR). When the mold is released from the wafer, the nanoscale pattern is left behind on the PR. The nanoscale features located on a nanoimprinting mold are patterned by direct nanolithographic techniques. Examples of direct nanolithography include electron beam direct-write and focused ion beam milling. Bulk and surface nanomachining can consistently produce 1D nanochannels, where the depth is normally the nanoscale dimension. Nanoimprint and direct nanolithography is known for being able to produce 2D nanochannels with well-defined channel walls [25]. Although these methods of nanochannel formation are viable, they require special tools and processes that are not widely available and/or they have negative drawbacks. One major drawback is the ability to align nanoscale electrodes along the nanochannel for sensing capabilities. The importance of having aligned nanoelectrodes along the nanochannel is discussed below. Previous research has demonstrated that an atomic force microscope (AFM) can be operated to successfully realize 2D nanochannels in silicon substrates [26, 27].
Atomic Force Microscopy: Atomic force microscopy (AFM) is typically used as an atomic scale surface profilometer and is a widely known machine in nanotechnology. Other tools, such as a scanning tunneling microscope (STM), scanning electron microscope (SEM) or a dektak surface profilometer are widely used for surface imaging in addition to an AFM. Unlike a SEM, where topographical images are generated by low angle surface imaging, an AFM generates topographical images based on data points obtained by physical vertical displacement of the tip and cantilever. An AFM is mainly composed of a silicon cantilever with a sharp tip fixed to the end. The tip is used to scan the topography of a surface, such as glass, ceramic, or biological samples. When the tip interacts with the surface, the cantilever deflects. This deflection is detected by a laser and photodiode configuration. The sample rests on a piezo scanner that contains a piezoelectric tube that can move the sample in the vertical direction and maintain a constant force on the sample. The data obtained by the cantilever deflection and photodiode is transferred into a high resolution image of the sample. There are several modes under which the AFM can operate. The four most common modes are contact, non-contact, dynamic mode, and force modulation mode. The most widely used mode in this research was contact mode for AFM nanolithography.
AFM Nanolithography: The manipulation of an AFM probe to scratch, indent, or remove a desired portion away from the surface of a substrate is known as AFM nanolithography. In general, AFM nanolithography can be categorized into two groups: bias-assisted AFM nanolithography and force-assisted AFM nanolithography. In the bias-assisted technique, the AFM tip is biased to create a localized electric field and acts as a nanoscale electrode for current injection or collection. Patterns can be formed as a result of electrostatic, electrochemical, field emission, and explosive gas discharge processes [28]. Force-assisted techniques were used in this research for 2D nanochannel realization. This method of AFM nanolithography has been studied and characterized by previous research under the guidance of Dr. Steve Tung [29]. This method consists of operating the tip in contact mode with an applied load on the sample surface. The tip is used to mechanically cut or scratch away the sample's surface to a desired pattern or nanochannel. The tip is pressed into the normal direction of the sample's surface area and moved in a straight line across the sample. Several parameters can be controlled during this process, including the force setpoint, tip speed, scratch direction, and number of cuts. AFM nanolithography was explored in the present research for nanochannel formation. The details of AFM nanolithography techniques used and correlation experiments completed in this work is discussed below.
Focused Ion Beam: In addition to AFM nanolithography, a focused ion beam (FIB) can also be used for nanochannel formation. An FIB is a nanotechnology tool that is normally coupled with a SEM for imaging purposes. A SEM is a microscope that uses electrons as opposed to light to produce high resolution images. Due to its multiple applications and nanoscale capabilities, the FIB is one of the most cutting edge pieces of equipment for nanotechnology research, with modern day resolution limits around 5-10 nm [30]. The major uses for the FIB are milling, deposition, implantation, and imaging. While the electron gun is used for surface imaging, the ion gun is the main source of making surface alterations since ions are much more massive than electrons. Gallium (Ga) is the most common ion used for FIB due to its high atomic weight of 69.723 g/mol and relatively small atomic radius of 1.35 Å [31]. Most ion beams use a liquid-metal ion source (LMIS) that are heated and accelerated downward to the sample under high electric field somewhere on the order of 108 V/cm while being held under a constant chamber pressure around 10−7 mbar. As a result of the electric field, the ions travel through the column components and are focused through the tip of the tungsten needle, known as the Taylor cone. The ions are funneled through this approximately 2 nm wide cone and bombarded towards the surface with any energy between 1-50 keV and a current between 1 pA-10 nA. The FIB column is normal to the sample surface. For ion milling or sputtering, Ga+ ions are accelerated towards the sample surface. During the sputtering process, secondary ions (+ or −) are removed from the surface as the beam of ions is raster scanned across the surface with a 11.5 nm pitch and 1 μs dwell time. Moreover, the incident ion beam produces secondary electrons. These secondary ions and electrons are detected and their signal produces the image of the sample's surface [32]. Thus, the FIB is a reliable and valid source of nanochannel formation in addition to AFM nanolithography.
In addition to FIB milling, nanoscale metal deposition is becoming increasingly significant in the field of nanotechnology. By using the same experimental setup as demonstrated in FIG. 6, metals can be deposited on the sample surface with nanoscale accuracy via FIB chemical vapor deposition [33]. The two most common metals commercially available for FIB maskless deposition are platinum (Pt) and tungsten (W). First, the gas injector must be initiated and brought within a few hundred micrometers of the sample surface. The desired gas is injected and absorbed onto the sample surface. Then, the Ga+ ions are accelerated into the surface and break the chemical bonds on the surface of the deposited gas. Dissociated molecules from this volatile reaction are desorbed from the surface and removed by vacuum, leaving behind the desired metal on the surface. It is notable to point out that the deposited metal is not 100% pure, mainly due to the fact that some Ga+ ions are implanted into the surface [32].
Potential Applications of Integrated Nanofluidic Systems: The FIB can be extremely beneficial for nanofluidic device fabrication. For rapid biomolecule detection, such as DNA nucleotides and avian influenza viruses (AIV), there must be a sensor embedded on the device so electrical measurements can serve as the detection element [34]. Due to the nanoscale resolution and capabilities of the FIB, making metal nanosensors is a definite possibility. For instance, biomolecules can be passed through a nanochannel surrounded by nanoelectrodes that serve as the sensing mechanism for the device. By combining nanoelectrodes with a transverse nanochannel, the biomolecules can be translocated through the nanofluidic system and sensed by the electrical sensors. Since some biomolecules, such as DNA, are negatively charged in nature, they can be suspended in a conductive carrying solution and driven through a nanochannel by applying an electric field across the channel. Meanwhile, the current signal across the nanoelectrodes can be monitored in real time as denoted by the double-headed red arrow in FIG. 7. This current that is measured is denoted as the tunneling current, for this is the current that flows across the backbone of each individual biomolecule. For DNA, each nucleotide (A, T, C, and G) has a unique electronic structure, the tunneling current will be different for each base and this can serve as the sequencing mechanism of the device. Previous experiments and theoretical calculations have been conducted to demonstrate that all four nucleobases exhibit unique electrical signatures [35, 36]. Scanning tunneling microscopy (STM) was used in references 35 and 36 cited herein to demonstrate the DNA nucleobase tunneling phenomenon and was validated by mathematical calculations using Green's function.
One potential drawback of a solid state device is the adjacent spacing of the nanoelectrodes. Since the inter-nucleotide spacing is only 0.34 nm for single stranded DNA (ssDNA), the nanoelectrodes must be fabricated on the sub-nanometer scale in order achieve single nucleotide detection [37]. However, surface chemistry techniques can be used to possibly functionalize electrodes and resolve the sub-nanometer electrode spacing problem. Today, there is a heavy international research effort to revolutionize current DNA sequencing methods by discovering a rapid, inexpensive, label-free method.